The extracellular neutral cysteine proteinase is a key virulence factor for Entamoeba histolytica. It degrades components of the extracellular matrix, including collagen, elastin, laminin, and fibronectin to aid in invasion. It may also have important interactions with the host immune system as demonstrated by our finding that the purified enzyme activates the alternative pathway of complement by cleaving C3. Additional evidence of the importance of the cysteine proteinase in the pathogenesis of amebiasis includes greater release of the proteinase by virulent axenic strains, the inability of proteinase negative mutants to injure cells in tissue culture, and the production of extracellular proteinase only by strains isolated from patients with invasive disease. Because there is no axenized nonpathogenic strain to allow comparative studies of virulence, we propose to study the structure and function of the proteinase in well-characterized clinical isolates of defined pathogenicity with the following specific aims: 1.) To compare the neutral cysteine proteinases of pathogenic and nonpathogenic clinical isolates of E. histolytica. These studies are designed to elucidate differences between the: a.) structure, b.) immunologic reactivity, and c.) substrate and inhibitor profiles of the cysteine proteinase(s) of pathogenic and nonpathogenic E. histolytica. 2.) To extend our initial observations of the interactions of the purified proteinase with the host's immune system by examining the ability of the proteinase to a.) release the anaphylatoxins C3a and C5a by specific cleavage of C3 and C5, b.) cleave IG=gA, and c.) cleave kininogen, liberating the vasodilatory peptide, bradykinin, which could promote vascular invasion. 3.) To determine if the same gene which encodes the neutral cysteine proteinase of pathogenic amebae is also present in nonpathogenic strains, and if so, determine if it is differentially expressed. These studies should provide an understanding of a key virulence factor of E. histolytica through investigations at the biochemical and molecular level in recent clinical isolates.